Ecotin
Ecotin is a previously characterized E. coli periplasmic protein known to inhibit the serine proteases; pancreatic trypsin (bovine), pancreatic chymotrypsin (bovine), and elastase (porcine) and has been postulated to play a role in protecting the bacteria from these exogenous proteases found in the mammalian gut (Chung, C. H. et al., J. Biol. Chem. 258:11032-11038 [1983]). It does not inhibit any known proteases from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, Pi, or the esterases "protease I and II"). It is further reported not to inhibit other mammalian serine proteases including; kallikrein, plasmin, and thrombin and non-mammalian proteases; papain(a sulfhydryl protease), pepsin(a carboxyl protease), subtilisin, and thermolysin(a metallo-protease) (Chung, C. H. et al., supra). McGrath et al. (McGrath, M. E. et al., J. Biol. Chem. 266:6620-6625 [1991]), report the cloning and sequencing of this 142 amino acid residue 16,096 Da inhibitor and that the scissile P.sub.1 -P.sub.1' bond is Met84-Met85 which lies within a disulfide-bonded loop formed by Cys50 and Cys87. These authors point out that, generally, the P.sub.1 residue of a particular serine protease inhibitor needs to be suitably matched with its protease so that favorable binding interactions can be generated between the P.sub.1 residue side chain and the substrate binding pocket of the protease. Thus given that trypsin favors the positively charged Lys and Arg at P.sub.1, chymotrypsin favors the large hydrophobic Tyr, Phe, Leu, and Met at P.sub.1, and elastase favors the small hydrophobic Leu and Val it is surprising that a single inhibitor like ecotin can strongly inhibit all three proteases. The ability of ecotin to inhibit these pancreatic proteases having such widely different P.sub.1 binding pockets has been attributed to the P.sub.1 Met residue which is apparently well tolerated by all three proteases while still generating significant binding energy. Other serine protease inhibitors having Met at P.sub.1 that show this pan-inhibition of the three pancreatic proteases include certain Kazal-type inhibitors and .alpha..sub.1 -proteinase inhibitor (sometimes referred to as .alpha..sub.1 -antitrypsin).
Serpins(serine protease inhibitors) such as .alpha..sub.1 -proteinase inhibitor have been well charcterized for their ability to inhibit various proteases because of their therapeutic potential to control proteolysis in thrombosis, shock, and inflammation (Schapira, M. et al., Trends Cardiovasc. Med., 4:146-151[1991]; Patston, P. A. et al., J. Biol. Chem. 265:10786-10791 [1990]) and because spontaneous mutations to the P.sub.1 residue (Met.sup.358 -Arg.sup.358 ; .alpha..sub.1 -proteinase inhibitor-Pittsburgh) dramatically alter the protease inhibitor specificity (Scott, C. F. et al., J. Clin. Invest. 77:631-634[1986]). .alpha..sub.1 -Proteinase inhibitor, having a Met residue at P.sub.1, is known to be a poor inhibitor of Factor Xa (Travis, J. et al., Biol. Chem. Hoppe-Seyler 367:853-859[1986]; see e.g. table page 857) as well as Factor XIIf (Scott, C. F. et al., supra; see Table I, page 632) and kallikrein (Scott, C. F. et al., supra; Schapira, M. et al., supra see Table 4, page 148). Thus from statements made in the literature and reasoning by analogy with other protease inhibitors having a P.sub.1 -Met, one would not predict that ecotin would be a good inhibitor of FXa, FXIIa or kallikrein.